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An accumulation of autochthonous cases of canine babesiosis caused by Babesia canis has been registered in a small animal clinic in the Saarland since the beginning of 2006, some cases with fatal outcome. This study aims to contribute to the explanation of strong focal occurrence of infections with B. canis in this region.Therefore, patient owners who had presented their dogs in the years 2006 and 2007 because of babesiosis and who had claimed not having left the Saarland with their dogs at least six months before the outbreak of Babesiosis, were asked for their dog walking habits. Accordingly, a selection often tick collection sites of various landscape structures was made.Tick sampling by flagging the vegetation took place every month from March to December 2008. The collected ticks were identified morphologically. In eight of ten collecting sites a total of 397 adult Dermacentor reticulatus ticks were collected from March to December with the highest frequencies during the months of May, October and November. All collected specimens were examined by genus-specific conventional PCR for the presence of Babesia-DNA. In positive samples, the PCR-products were differentiated by sequencing. ten D. reticulatus (2.5%) ticks examined were found positive for DNA of B. canis canis originating from three out of eight collection sites. Consequently, an endemic distribution of D. reticulatus was confirmed and a natural

Neospora (N.) caninum is a protozoan parasite which is regarded as a major cause of abortion in cattle. Dogs and coyotes are definitive hosts of N. caninum which may shed environmentally resistant stages, oocysts, in their feces. Epidemiological studies in Germany showed that the presence of dogs increased the risk of a bovine herd to be N. caninum-positive in a bulk-milk ELISA test. However, there were also N. caninum-positive herds where dogs were not kept together with cattle.This leads to the question whether canids other than dogs, e.g., foxes, might be involved in the horizontal transmission of N. caninum. Therefore, the aim of our examinations in wild animals was to find out whether there are indications for a sylvatic cycle with foxes as definitive hosts and deer, roe deer and wild mice samples contained structures which resembled those of coccidian oocysts. In 13 of these 65 samples coccidian DNA was detected using a 18S rRNA gene based polymerase chain reaction (PCR).The examination of the 65 samples in a N. caninum-specific PCR revealed no positive result. Hammondia (H.) heydorni-DNA was detected in two samples. In addition, brain samples from 528 foxes, 224 wild mice, 16 deer and roe deer as well as from 1 wild boar were examined for the presence of N. caninum DNA by real time PCR. All samples tested negative by PCR. In conclusion, our study yielded no evidence indicating that the examined animals were part of a sylvatic cycle for N. caninum.

Suspected of having a systemic malignancy a 22-month-old boy was admitted to hospital with fever, pancytopenia and hepatosplenomegaly. The boy was of ethnically German origin and no travel abroad was reported.Intensive search for a focus of infection, laboratory tests and bone marrow microscopy failed to be diagnostic. Serological findings and detection of Leishmania DNA in bone marrow by polymerase chain reaction (PCR) led to the diagnosis of visceral leishmaniasis. On explicit questioning the child's parents reported a stay in Greece 18 months before onset of symptoms.On the fourth day of i.v. therapy with liposomal amphotericin B, 3mg/kg/d for 10 days, the fever subsided. Platelets and leukocytes regained normal levels. The child was discharged after 10 days of treatment and received two more doses on days 14 and 21.Negative results on microscopic bone marrow inspection do not rule out visceral leishmaniasis. Detection of anti-Leishmania antibodies may support the suspected diagnosis and provide the indication for PCR technique.

Clinical standards to confirm babesiosis in dogs include the direct identification of the infectious agent in blood smears and serological assays for Babesia canis-specific antibodies. Here, we demonstrate in seven cases (with data on anamnesis, clinics, laboratory diagnostics, and therapeutic outcomes) that a new diagnostic procedure is required. This is the molecular-genetic identification of babesia by real time PCR allowing an unequivocal identification of the infectious agents. Indeed, all seven patients presenting severe clinical symptoms were PCR-positive, but only two of them had specific antibodies and showed babesia in their bloodstream. Six of the dogs appeared to have acquired babesiosis while travelling abroad, and one in the Swiss canton of Schaffhausen.

Neospora caninum represents one of the most frequent abortifaciant organisms worldwide. The parasite is diaplacentally transmitted from the pregnant cow to the fetus, where it normally leads to the delivery of a healthy, however persistently infected calf. Abortion thus is a relative rare event. The transmission of bovine neosporosis occurs in more than 90% of the cases vertically due to the endogenous reactivation of a persistently infected mother. Exogenous infections are therefore responsible for less than 10% of the cases. The question arises about which infection sources may be relevant in this context. In Switzerland, the role of dogs as definitive hosts has been shown to be of low significance in that respect. Recently, discussion focused on the potential of infectious bull semen following natural or artificial insemination. Thus, a few years ago a report documented the detectability of N. caninum-DNA in the semen of naturally infected bulls by nested-PCR. As a consequence, we decided to gain own experience by investigating 5 separate semen specimens per animal, originating from 20 N. caninum-seropositive bulls used for artificial insemination in Switzerland. All probes turned out to be negative by nested PCR. Based upon our laboratory experiences, the potential bull semen-associated Neospora-problem seems not to affect the Swiss bull population, thus there is no evidence to include further respective means of control.

Starting in November 2003 a series of five clinical cases of canine babesiosis was registered in the region of Obergösgen (canton Solothurn). All presented dogs showed increased body temperature, thrombocytopenia and hemoglobinuria, and none of the dogs had been abroad or visited endemic regions in the southern or western part of Switzerland so far. Babesia canis was detected in the blood smears of all five patients, but only three had detectable specific antibodies against this parasite. However, seroconversion was found in a second sample collected from the negative dogs at a later timepoint, confirming the diagnosis of canine babesiosis. The blood samples of two parasitized dogs were used for DNA-isolation and were tested with a Babesia-specific PCR, detecting the 18S rRNA-gene. Sequencing of the amplified products revealed a 100% identity with the sub-species B. canis canis. The ticks Rhipicephalus sanguineus and Dermacentor marginatus are potential vectors for B. canis. In the area where the infection with B. canis was suspected a total of 152 ticks was collected and characterized; one was a female R. sanguineus.Although babesia could not be detected in the latter tick and the final prooffor the complete life cycle is still lacking, it is very probable that B. canis has become autochthonous in the canton Solothurn.

Leishmania spp. are the causative agents of visceral, cutaneous and mucocutaneous leishmaniosis with several taxa ("species") of the genus Leishmania being involved in human disease. As diagnostics based on microscopical detection of the parasites or on serological tests are often unsatisfactory, also molecular biological methods, particularly the polymerase chain reaction (PCR), have been employed for the detection of Leishmania spp. in the past years. The aim of the present study was to compare different PCR-protocols and optimise them for our needs, placing emphasis on the improvement of DNA isolation. PCR was performed with whole cell DNA isolated from cultures, as well as from simulated blood samples and clinical samples. Three different methods for the isolation of DNA from blood samples and two different PCR-protocols, one amplifying a fragment of the 18S rDNA and one for the amplification of the whole kDNA-circle, were applied and compared. No significant difference in sensitivity was detected between the different PCR-protocols, however, it was shown that the highest yield of DNA was achieved with a DNA isolation protocol based on urea.

Three weeks after returning from a trip to Guatemala, a 33-year-old man developed two ulcers with indurated edges on his right leg and painful lymph nodes in the right groin. His general condition was not impaired.Histological examination revealed cellular infiltrates of the corium by lymphocytes and plasma cells, always accompanied by epithelial cells and multinuclear giant cells. Special stainings were unable to detect pathogens but Leishmania brasiliensis was identified using PCR. The Leishmania culture remained negative.After 7-day intravenous therapy with 20 mg/kg/d pentostam (pentavalent antimonial compound), the patient developed gastrointestinal complaints, coupled with a marked elevation of transaminases. Therapy was discontinued until the transaminase values normalized, then continued in reduced dosage (12 mg/kg body weight) for 23 days. The ulcers and lymphadenitis healed under this therapy.The diagnosis of American cutaneous Leishmaniasis may be complicated by the relative lack of pathogens in the lesions. PCR diagnosis are very helpful here. The therapy must be systemic owing to the danger of progression to mucocutaneous Leishmaniasis. The standard therapeutic pentostam has, however, a high rate of side effects and administration is exclusively intravenous.

Leishmaniasis is an anthropozoonosis caused by infection with leishmania parasites with either cutaneous, mucosal or visceral (kala-azar) involvement. While the benign cutaneous form is self-limited death occurs in approximately 80% of children with kala-azar when untreated. The diagnosis of kala-azar should not be missed in children presenting with fever, hepatosplenomegaly and pancytopenia especially with a history of sand fly bites. We report the case of a 13-month-old boy with both cutaneous and visceral involvement.

Cyst-forming coccidia may cause significant losses in livestock, primarily due to abortion, loss of young animals and neuromuscular diseases. Rather recently, Neospora caninum has been recognized as one of the major protozoal abortion-inducing parasites in cattle. The present study addressed the performance of different diagnostic tools (in vitro-cultivation; histology; immunohistochemistry; serology; PCR) suitable for the direct or indirect detection of N. caninum. By PCR, Neospora-DNA was detected in 24 brains (29%) from 83 bovine abortion, many of these brains were simultaneously characterized by histopathological findings typical for a protozoal, cerebral parasitosis. The diagnostic methods were furthermore assessed using samples of different tissues and body fluids from three experimentally Neospora-infected pregnant cows and their foetuses. The diaplacental passage of N. caninum to the foetus was successful in two of the three cases. In these two cases, PCR was positive for different foetal organs and, additionally, for the abomasal and amniotic fluid. The successfully infected cows developed anti-Neospora serum antibodies between 10 and 17 days post infection, foetuses remained serologically negative in all cases. The results obtained in the present study demonstrated the usefulness of PCR, complemented by serology, for the specific diagnosis of bovine neosporosis. Such tests may prove suitable to perform epidemiological investigations. Taken together, our data indicated that prenatal neosporosis may be an important cause of infectious bovine abortion in Switzerland.

A polymerase chain reaction was applied to detect Toxoplasma gondii DNA in placental and fetal tissue samples of 47 unselected ovine abortions of the lambing season 1990/91 (Baden-Württemberg, Rhineland, Hesse). For the amplification a 190 bp or 223 bp sequence of the B1-gene of T. gondii was selected as the target sequence. Both sequences were detected in five abortions. All positive results were immunohistochemically confirmed using the peroxidase antiperoxidase technique (PAP-staining). Thus, in Germany, too, T. gondii infection in sheep during pregnancy should be considered as a possible cause of abortions, particularly in case of abortions of unknown genesis.

Primary infection with Toxoplasma gondii during pregnancy may affect the fetus and result in congenital toxoplasmosis. In Austria serological screening for detection of newly acquired infection during pregnancy was introduced in 1975. In this study we used polymerase chain reaction (PCR) for detection of fetal infection with Toxoplasma gondii. Amniotic fluid samples were analyzed from 11 women with serological indication of acute toxoplasmosis infection. Nine of these women had already received treatment prior to amnio-centesis and no evidence of Toxoplasma gondii DNA was detected with PCR in the respective amniotic fluid samples. Isolation of the organism by mouse inoculation was negative in these cases and follow-up serology as well as clinical examination of the infants confirmed these results. In 2 patients investigation of the amniotic fluid samples by means of PCR was positive; both women had not yet been treated at the time of amniocentesis. Our results indicate that identification of Toxoplasma gondii in amniotic fluid is a useful procedure for diagnosing or excluding fetal infection. Moreover, the current recommendations of the screening program appear to be successful in preventing congenital toxoplasmosis.